Abstract:

The present invention is concerned with an antigenic composition
comprising at least one antigen that comprises at least one antigenic
epitope or antigenic determinant derived from a protein present in one or
both of S. equi subsp. equi and subsp. zooepidemicus and use thereof for
immunization of non-human mammals against S. equi subsp. equi and/or
subsp. zooepidemicus. The present invention also discloses a vaccine
composition comprising the aforesaid antigenic composition as immunizing
component.

Claims:

1. An antigenic composition comprising at least one antigen, wherein said
at least one antigen comprises at least part of a protein or polypeptide
of Streptococcus equi subsp. equi or subsp. zooepidemicus and said at
least part of said protein or polypeptide comprises at least one
antigenic epitope or antigenic determinant of Streptococcus equi, and
wherein said protein or polypeptide is selected from the group
comprisinga protein or polypeptide which is designated EAG and has an
amino acid sequence as shown in SEQ ID NO: 13;a protein or polypeptide
which is designated IdeE and has an amino acid sequence as shown in SEQ
ID NO: 10;a protein or polypeptide which is designated IdeE2 and has an
amino acid sequence as shown in SEQ ID NO: 1;a protein or polypeptide
which is designated Eq5 and has an amino acid sequence as shown in SEQ ID
NO: 3;a protein or polypeptide which is designated Eq8 and has an amino
acid sequence as shown in SEQ ID NO: 5;a protein or polypeptide which is
designated IdeZ2 and has an amino acid sequence as shown in SEQ ID NO:
7;a protein or polypeptide which is designated Eqz5 and has an amino acid
sequence as shown in SEQ ID NO: 8; anda protein or polypeptide which is
designated Eqz8 and has an amino acid sequence as shown in SEQ ID NO:
9;or an analog or fragment thereof, and wherein a composition which
comprises EAG comprises at least one further antigen, which is a protein
or polypeptide which is selected from the group comprising IdeE, IdeE2,
Eq5, Eq8, IdeZ2, Eqz5 and Eqz8.

2. The antigenic composition of claim 1, wherein said at least one protein
or polypeptide is selected from the group consisting of IdeE and IdeE2
and wherein a composition that comprises IdeE2 comprises at least one
further antigen.

3. The antigenic composition of claim 1 or 2, wherein said at least one
protein or polypeptide is selected from the group consisting of Eq5 and
Eq8.

4. The antigenic composition of claim 1, wherein said at least one protein
or polypeptide is selected from the group consisting of EAG, Eq5 and Eq8
and which composition further comprises at least one antigen, which is
selected from the group comprising a protein or a polypeptide designated
CNE (or SEC), which has an amino acid sequence as shown in SEQ ID NO: 28,
and a protein or a polypeptide designated ScIC, which has an amino acid
sequence as shown in SEQ ID NO: 29.

5. The antigenic composition of claim 4, wherein said at least one protein
or polypeptide is selected from the group comprising IdeE and IdeE2.

7. The antigenic composition of claim 1, wherein said at least one protein
or polypeptide is selected from the group consisting of EAG, Eq8, and
IdeE2 and which composition comprises at least one further antigen which
is selected from the group comprising IdeE, Eq5, IdeZ2, Eqz5 and Eqz8.

8. The antigenic composition of claim 1, wherein at least one antigen is
recombinantly produced.

9. The antigenic composition of claim 1, wherein at least one antigen is
an isolated or purified antigen.

10. The antigenic composition of claim 1, which comprises at least one
recombinant vector and at least one polynucleotide inserted therein that
encodes said at least one protein or polypeptide, andwhich vector is able
to express said polypeptide in vivo in a non-human mammal susceptible to
infection with S. equi.

11. The antigenic composition of claim 10, wherein the vector is an
expression vector which is a plasmid or a viral vector and wherein said
polynucleotide has a nucleotide sequence as shown in SEQ ID NOS: 12 and
14-21.

12. The antigenic composition of claim 1, which is an immunogenic
composition.

13. A vaccine composition for protecting non-human mammals against
infection of Streptococcus equi, which comprises the antigenic
composition of claim 1 as immunizing component, and a pharmaceutically
acceptable carrier.

14. The vaccine composition of claim 13, which further comprises an
adjuvant.

15. The vaccine composition of claim 13, which is a vaccine that protects
susceptible mammals, suitably horses, against strangles caused by
Streptococcus equi subsp. equi.

16. The vaccine composition of claim 13, which is provided in a
physiologically administrable form, and suitably is administrable by
subcutaneous or intranasal inoculation.

18. A method for producing an antigen or an immunogen of an antigenic
composition of claim 1, which method comprises(a) providing a DNA
fragment encoding said antigen and introducing said fragment into an
expression vector;(b) introducing said vector, which contains said DNA
fragment, into a compatible host cell;(c) culturing said host cell
provided in step (b) under conditions required for expression of the
product encoded by said DNA fragment; and(d) isolating the expressed
product from the cultured host cell, and, optionally,(e) purifying the
isolated product from step (d) by affinity chromatography or other
chromatographic methods known in the art.

19. A method for preparation of a vaccine composition according to claim
13, which vaccine composition contains as immunizing component, said
antigenic or immunogenic composition, said method comprising mixing said
antigenic or immunogenic composition and a pharmaceutically acceptable
carrier.

20. Use of an antigenic or immunogenic composition of claim 1 in the
preparation of a vaccine protecting against S. equi infection inclusive
of strangles caused by subsp. equi infection in horses.

21. A method for the production of an antiserum, said method comprising
administering an antigenic preparation of claim 1 to an animal host to
produce antibodies in said animal host and recovering antiserum
containing said antibodies produced in said animal host.

22. A method of prophylactic or therapeutic treatment of S. equi infection
in non-human mammals, suitably horses, comprising administering to said
mammal an immunologically effective amount of a vaccine composition of
claim 13 or an antiserum produced by administering an antigenic
composition to an animal host to produce antibodies in said animal host
and recovering antiserum containing said antibodies produced in said
animal host,wherein said antigenic composition comprises at least one
antigen, wherein said at least one antigen comprises at least part of a
protein or polypeptide of Streptococcus equi subsp. equi or subsp.
zooepidemicus and said at least part of said protein or polypeptide
comprises at least one antigenic epitope or antigenic determinant of
Streptococcus equi, and wherein said protein or polypeptide is selected
from the group comprisinga protein or polypeptide which is designated EAG
and has an amino acid sequence as shown in SEQ ID NO: 13;a protein or
polypeptide which is designated IdeE and has an amino acid sequence as
shown in SEQ ID NO: 10;a protein or polypeptide which is designated IdeE2
and has an amino acid sequence as shown in SEQ ID NO: 1;a protein or
polypeptide which is designated Eq5 and has an amino acid sequence as
shown in SEQ ID NO: 3;a protein or polypeptide which is designated Eq8
and has an amino acid sequence as shown in SEQ ID NO: 5;a protein or
polypeptide which is designated IdeZ2 and has an amino acid sequence as
shown in SEQ ID NO: 7;a protein or polypeptide which is designated Eqz5
and has an amino acid sequence as shown in SEQ ID NO: 8; anda protein or
polypeptide which is designated Eqz8 and has an amino acid sequence as
shown in SEQ ID NO: 9;or an analog or fragment thereof, and wherein a
composition which comprises EAG comprises at least one further antigen,
which is a protein or polypeptide which is selected from the group
comprising IdeE, IdeE2, Eq5, Eq8, IdeZ2, Eqz5 and Eqz8.

23. A method for protecting horses against Streptococcus equi infection,
which comprises inoculating a horse subcutaneously or intranasally with a
vaccine composition of claim 13 to induce an immune response against
Streptococcus equi in said horse.

24. The method of claim 23, wherein an immune response in the form of IgG
and/or IgA and/or IgM antibodies in the nasopharyngeal mucus is induced
in said horse.

25. An antibody preparation comprising at least one, and suitably at least
two, antibodies specific for a protein or polypeptide of the composition
of claim 1, which antibody/antibodies is/are polyclonal or monoclonal; or
which preparation comprises a fragment of said antibodies.

26. The antibody preparation of claim 25 which is used prophylactically or
therapeutically against strangles and provides passive immunization when
administered to a non-human mammal susceptible to infection by
Streptococcus equi or infected by Streptococcus equi.

Description:

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002]This invention is generally related to antigenic or immunogenic
compositions and use thereof for immunization of non-human mammals, e.g.
horses, against Streptococcus equi.

[0003]2. Background of the Invention

[0004]Streptococcal infections in horses are mainly caused by the species
Streptococcus equi, which is classified as a Lancefield Group C
Streptococcus and comprises two subspecies designated equi and
zooepidemicus, respectively.

[0005]Streptococcus equi subsp. equi, which is virtually confined to
horses, is the causative agent of strangles, a world-wide distributed and
highly contagious serious disease of the upper respiratory tract of the
Equidae. Strangles is one of the most frequently reported equine diseases
world-wide and is characterized by fever, nasal discharge, and abscess
formation in the retropharyngeal and mandibular lymph nodes. In some
cases the disease shows a metastatic course in the body, so called
"bastard strangles". The disease has a world-wide distribution and causes
great economic losses. Moreover, since strangles is a highly contagious
disease, not only infected animals but also all other members of e.g. an
afflicted stud must be isolated for as long as up to three months.

[0006]S. equi subsp. zooepidemicus is considered as an opportunistic
commensal often occurring in the upper respiratory tract of healthy
horses. However, after stress or virus infection, it can cause a
secondary infection, which results in strangles-like symptoms. Moreover,
subsp. zooepidemicus infects not only horses but also a wide range of
other animals, like pigs, dogs, cats, and cows. Even human cases of
infection due to subsp. zooepidemicus have been reported. This subspecies
has been implicated as the primary pathogen in conditions such as
endometritis, cervicitis, abortion, mastitis, pneumonia, abscesses and
joint infections.

[0007]Although it is possible to treat and cure these streptococcal
infections with antibiotics, such as penicillin, tetracycline or
gentamicin, an effective prophylactic agent that could prevent outbursts
of such infections and obviate or reduce the risk for development of
resistant strains associated with antibiotic treatment, would be
appreciated.

[0008]3. Description of the Related Art

[0009]However, although many attempts have been made to develop
prophylactic agents such as vaccines against S. equi, at the present time
no efficient vaccines or immunizing preparations are available, neither
for the subspecies equi nor for the subspecies zooepidemicus.

[0010]Existing vaccines against strangles are based on inactivated, e.g.
heat-killed, or attenuated strains of S. equi subsp. equi or acid
extracts/mutanolysin enriched in M-protein(s), i.e. immunogenic
protein(s) produced by S. equi. A vaccine against S. equi subsp.
zooepidemicus based on an M-like protein is disclosed in U.S. Pat. No.
5,583,014. In WO 87/00436, an avirulent strain of S. equi is disclosed
for use as a vaccine against S. equi that stimulates an antibody response
in the nasopharyngeal mucosa after administration thereof to a horse.

Recently, a commercial vaccine against strangles, Equilis StrepE from
IntervetVET, UK, has been released in Great Britain (November 2004),
which vaccine also has been used throughout Europe and in South Africa
and South America. However, the safety and efficacy of this vaccine,
which is based on an attenuated (living, deletion mutated) strain of S.
equi subsp. equi, can be questioned.

[0011]Since the previously developed vaccines or immunizing preparations
are hampered by side-effects and, moreover, provide insufficient
protection, there is a need for efficient and safe prophylactic agents,
such as vaccines, that protect against S. equi infections and/or prevent
spread thereof without giving rise to undesirable side-effects.

[0012]It is well known that attachment to eukaryotic cell surfaces is an
essential step in the establishment of infection and colonization by
bacterial pathogens. Accordingly, streptococcal surface proteins, that
interact with and/or bind to different components of the Extracellular
Matrix (ECM) or plasma proteins of the host cell, are potential
candidates for use as active component(s) for immunizing purposes.

[0013]This is illustrated by the vaccines based on M-like proteins
mentioned above or disclosed in the literature, i.a. in WO 98/01561. The
binding of fibrinogen and complement factor H to M-proteins is assumed to
be important for the ability of streptococci to resist phagocytosis.

[0014]Another mechanism used by streptococci for attachment to host cells
involves binding to the ECM component fibronectin (Fn) (Ref. 21, 22).
Binding between Fn-binding bacterial cell-surface proteins and
immobilized Fn promotes internalization of streptococci by epithelial
cells (Ref. 2, 23, 24). Fibronectin is a dimeric glycoprotein found both
in plasma and in a fibrillar form in the extracellular matrix. The main
function of Fn is to mediate substrate adhesion of eukaryotic cells,
which involves the binding of specific cell-surface receptors to certain
domains of the Fn molecule. Furthermore, it also interacts with several
other macromolecules, such as DNA, heparin, fibrin, and collagen.

[0015]Accordingly, Fn-binding proteins from different streptococcal
species have been cloned and sequenced previously. For instance, from S.
equi, one Fn-binding protein has been cloned and characterized, which is
a Fn-binding cell-surface protein of subsp. zooepidemicus, that has been
designated FNZ (Lindmark et al., 1996, Ref. 9). Another Fn-binding
protein from S. equi subsp. equi, has been cloned and characterized by
Lindmark and Guss (1999) (Ref. 12). This latter protein that is
designated SFS and its potential use as an active component in a vaccine
for protection of horses against strangles are disclosed in WO 00/37496.

[0016]In Jonsson et al. (1995) (Ref. 8), a protein designated ZAG has been
cloned and characterized from S. equi subsp. zooepidemicus that mediates
binding to the plasma proteinase inhibitor α2M. It is
speculated therein that this protein is similar in function to
streptococcal M proteins. This protein, ZAG, is also disclosed in WO
95/07296, where its α2M-binding properties are indicated.
However, immunogenic properties or potential use thereof as an active
component in a vaccine for protection of e.g. horses against strangles
are not disclosed therein. The gene zag encoding ZAG is also disclosed in
these references.

[0017]A gene that is similar to the aforesaid zag gene from S. equi subsp.
zooepidemicus but is present in subsp. equi has been described by
Lindmark et al. (1999) (Ref. 11) and Lindmark (1999) (Ref. 13). This gene
is hereafter designated eag and encodes a protein designated EAG.

[0018]In WO 2004/032957 A1, antigenic compositions are disclosed which
comprise at least one antigen derived from a protein designated EAG,
which protein is present in S. equi, and which composition suitably
comprises at least one further antigen selected from a group of proteins
which are present in S. equi and are designated FNZ, SFS, SEC and ScIC,
respectively.

[0019]In WO 2007/115059 A2, subunit immunogenic or vaccine compositions
are disclosed which comprise at least one polypeptide of S. equi having a
specific amino acid sequence as shown in the sequence listing attached to
said publication or an analog thereof or a fragment thereof which is a
part of said polypeptide and contains at least one epitope. However, no
results as regards immunizing of horses against strangles are provided in
this document.

[0020]In the study reported in Lannergard, J., Frykberg, L. and Guss, B.
(2003) FEMS Microbiol Lett 222: 69-74, (Ref. 28), a new gene designated
cne has been isolated and the corresponding protein CNE has been
characterized.

[0021]In Flock, M., Jacobsson, K., Frykberg, L., Hirst, T., R., Franklin,
A., Guss, B. and Flock, J.-I. (2004) Infect Immun 72:3228-3236 (Ref. 5),
it is reported that in a mouse model of equine strangles, parts of the
proteins designated FNZ, SFS and EAG, respectively, were used to immunize
mice. FNZ and EAG were considered as promising candidates for development
of a safe and efficacious vaccine against strangles.

[0023]In Vaccine (Timoney et al.; 2007) it is reported that a great number
of recombinant extracellular proteins of S. equi, including CNE (also
designated SEC) and Se 44.2 (also designated IdeE2) are useless as
vaccine components. It is speculated therein that earlier results for
SEC/CNE obtained for mice are not applicable to horses. Thus, it is not
obvious that recombinant forms of surface localized proteins necessarily
are likely candidates for vaccine components.

[0025]Although many efforts have been made to develop efficient vaccines
and some of the immunizing components of WO 2004/032957 A1 are promising
candidates for use in a vaccine that protects against S. equi infection,
development of safe vaccines having a high degree of immunogenicity and
exhibiting limited or no side effects is still desirable.

BRIEF SUMMARY OF THE INVENTION

[0026]The present invention is based on an antigenic, suitably an
immunogenic, composition comprising at least one antigen, suitably an
immunogen, that comprises at least one antigenic epitope or antigenic
determinant derived from a protein present in one or both of S. equi
subsp. equi and subsp. zooepidemicus and use thereof for immunization of
non-human mammals against S. equi subsp. equi and/or subsp.
zooepidemicus.

[0027]The present invention is also directed to a vaccine composition
comprising the afore-said antigenic composition as immunizing component;
to methods to prepare said antigenic, suitably immunogenic, composition
or vaccine composition; to methods to induce an immune response against
S. equi in non-human mammals; and to methods for prophylactic or
therapeutic treatment of S. equi infection in non-human mammals. When
used generally, the expression "S. equi" refers to one or both of subsp.
equi and subsp. zooepidemicus.

[0028]According to a suitable embodiment, the present invention is
directed to a vaccine that protects equines, such as horses, against
strangles.

[0029]In the context of infections caused by S. equi subsp. equi, the
expression "non-human mammals" primarily refers to animals belonging to
the family Equidae that consists of horses, donkeys and zebras and to
hybrids thereof, such as mules and hinnies. Camels and dromedaries are
also encompassed therein.

[0030]In connection with infections caused by S. equi subsp.
zooepidemicus, the expression "non-human mammals" in addition refers also
to other mammals such as cows, pigs, dogs and cats.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]In the following, the present invention is described in closer
detail with reference to the drawings, where:

[0051]FIG. 19 shows ELISA measurements of IgG antibodies in nasal washings
of seven immunized horses. The log dilution of sera required to give an
absorbance value at a cut-off of 1.0 was calculated for each individual
nasal wash sample. Mean values (n=7) with standard errors are shown.
Samples taken before (pre imm. day 1) and twelve days after the third
immunization are shown (day 86). The horses were immunized with EAG, CNE
and ScIC.

[0052]FIG. 20 shows ELISA measurements of IgG antibodies in sera of seven
immunized horses. The log dilution of sera required to give an absorbance
value at a cut-off of 1.5 was calculated for each individual serum
sample. Mean values (n=7) with standard errors are shown. Sample taken
before (day 1), after V2 (day71), and after V3 (day 86) are shown.

[0053]FIG. 21 shows ELISA measurements of IgG antibodies in sera of
immunized horses (Pentavac). The log dilution of sera required to give an
absorbance value at a cut-off of 1.5 was calculated for each individual
serum sample. Mean values (n=7) with standard errors are shown. Sample
taken before (day 1), after V2 (day 71), and after V3 (day 86) and
samples taken between V3 and V4 (day 270) are shown.

[0060]SEQ ID NO 7 shows the amino acid sequence of the protein IdeZ2 from
subsp. zooepidemicus.

[0061]SEQ ID NO 8 shows the amino acid sequence of the protein Eqz5 from
subsp. zooepidemicus.

[0062]SEQ ID NO 9 shows the amino acid sequence of the protein Eqz8 from
subsp. zooepidemicus.

[0063]SEQ ID NO 10 shows the amino acid sequence of the protein IdeE.

[0064]SEQ ID NO 11 shows the amino acid sequence of the protein IdeZ from
subsp. zooepidemicus.

[0065]SEQ ID NOS 12 and 13 shows, respectively, the nucleotide sequence of
the gene eag and the amino acid sequence of the protein EAG4B, which
protein is usually designated EAG in connection with the present
invention.

[0066]SEQ ID NO 14 shows the nucleotide sequence of the gene ideE2.

[0067]SEQ ID NO 15 shows the nucleotide sequence of the gene eq5.

[0068]SEQ ID NO 16 shows the nucleotide sequence of the gene eq8.

[0069]SEQ ID NO 17 shows the nucleotide sequence of the gene IdeZ2 from
subsp. zooepidemicus.

[0070]SEQ ID NO 18 shows the nucleotide sequence of the gene eqz5 from
subsp. zooepidemicus.

[0071]SEQ ID NO 19 shows the nucleotide sequence of the gene eqz8 from
subsp. zooepidemicus.

[0072]SEQ ID NO 20 shows the nucleotide sequence of the gene ideE.

[0073]SEQ ID NO 21 shows the nucleotide sequence of the gene ideZ from
subsp. zooepidemicus.

[0077]SEQ ID NO 30 shows the amino acid sequence of the recombinant IdeE
used for immunization.

[0078]SEQ ID NO 31-32 shows the nucleotide sequence of primers.

DETAILED DESCRIPTION OF THE INVENTION

[0079]The present invention is concerned with identification of
polypeptides or proteins of S. equi that are able to elicit an antigenic,
suitably an immunogenic, response, when administered to a non-human
mammal; and to the identification of polynucleotides or genes encoding
these polypeptides or proteins.

[0080]The present invention is also concerned with fragments or analogs of
said polypeptides or proteins or of said polynucleotides or genes.

[0081]More specifically, genes of S. equi encoding extracellular proteins
were identified and, subsequently, the corresponding products were
expressed and evaluated in vaccine studies. The present invention is at
least partly based on such studies.

[0082]Accordingly, the present invention relates to an antigenic
composition comprising at least one antigen, wherein said at least one
antigen comprises at least part of a protein of Streptococcus equi subsp.
equi or subsp. zooepidemicus, and said at least part of said protein
comprises at least one antigenic epitope or antigenic determinant of
Streptococcus equi.

[0083]According to one embodiment, the present invention is directed to an
antigenic composition comprising at least one antigen, wherein said at
least one antigen comprises at least part of a protein or polypeptide of
Streptococcus equi subsp. equi or subsp. zooepidemicus and said at least
part of said protein or polypeptide comprises at least one antigenic
epitope or antigenic determinant of Streptococcus equi, and wherein said
protein or polypeptide is selected from the group comprising:

[0084]a protein or polypeptide which is designated EAG and has an amino
acid sequence as shown in SEQ ID NO: 13;

[0085]a protein or polypeptide which is designated IdeE and has an amino
acid sequence as shown in SEQ ID NO: 10;

[0086]a protein or polypeptide which is designated IdeE2 and has an amino
acid sequence as shown in SEQ ID NO: 1;

[0087]a protein or polypeptide which is designated Eq5 and has an amino
acid sequence as shown in SEQ ID NO: 3;

[0088]a protein or polypeptide which is designated Eq8 and has an amino
acid sequence as shown in SEQ ID NO: 5;

[0089]a protein or polypeptide which is designated IdeZ2 and has an amino
acid sequence as shown in SEQ ID NO: 7;

[0090]a protein or polypeptide which is designated Eqz5 and has an amino
acid sequence as shown in SEQ ID NO: 8; and

[0091]a protein or polypeptide which is designated Eqz8 and has an amino
acid sequence as shown in SEQ ID NO: 9;

or an analog or a fragment thereof, and wherein a composition which
comprises EAG, comprises at least one further antigen, which is a protein
or polypeptide, which is selected from the group comprising IdeE, IdeE2,
Eq5, Eq8, IdeZ2, Eqz5, and Eqz8.

[0092]For convenience, the polypeptides having amino acid sequences as
shown in the sequence listing are frequently only designated EAG, IdeE,
IdeE2, Eq5, Eq8, IdeZ2, Eqz5, and Eqz8, respectively. EAG, IdeE, IdeE2,
Eq5, and Eq8 designate proteins that can be found in S. equi subsp. equi
and IdeZ, IdeZ2, Eqz5, and Eqz8 designate proteins that can be found in
S. equi subsp. zooepidemicus.

[0093]The antigens or immunogens of the present antigenic or immunogenic
compositions may comprise the entire amino acid sequence of said protein
or polypeptide or may comprise a fragment, e.g. a C-terminal or
N-terminal fragment thereof, or an analog thereof. For instance, an
N-terminal fragment of EAG is used according to various embodiments of
the present invention.

[0094]According to one embodiment, the present invention is related to an
antigenic or immunogenic composition which contains at least 2 or 3
antigens or immunogens selected from the group consisting of EAG, IdeE,
IdeE2, Eq5, Eq8, IdeZ, IdeZ2, Eqz5, and Eqz8.

[0095]According to a specific embodiment, the present invention is related
to an antigenic or immunogenic composition which contains at least 2 or 3
antigens or immunogens selected from the group consisting of EAG, IdeE,
IdeE2, Eq5, and Eq8. Suitably this composition also comprises one or both
of the previously described antigens ScIC (SEQ ID NO: 29) and CNE (SEQ ID
NO: 28) (also designated SEC e.g. SEC 2.16). A further embodiment is
related to an antigenic composition comprising EAG, ScIC, CNE, Eq5, and
Eq8.

[0096]A suitable composition contains 2 antigens or immunogens which are
comprised of Eq5 and Eq8, respectively. According to a further
embodiment, the present invention is directed to a composition that
contains 3 antigens or immunogens, which suitably are comprised of EAG,
IdeE, and IdeE2. The present invention is also related to compositions
that comprise one or both of IdeE and IdeE2.

[0097]The present invention is also related to an antigenic composition,
wherein said at least one protein or polypeptide is selected from the
group consisting of EAG, Eq5 and Eq8 and which composition further
comprises at least one antigen, which is selected from the group
comprising a protein or a polypeptide designated CNE (or SEC), which has
an amino acid sequence as shown in SEQ ID NO: 28, and a protein or a
polypeptide designated ScIC, which has an amino acid sequence as shown in
SEQ ID NO: 29. Suitably, said at least one protein or polypeptide is
selected from the group comprising IdeE and IdeE2.

[0098]Antigenic compositions of the present invention, which have been
shown to be useful in vaccine compositions, comprise according to one
embodiment, the antigens EAG, ScIC, CNE (or SEC), Eq5, Eq8, IdeE and
IdeE2, and according to another embodiment, the antigens EAG, ScIC, CNE
(or SEC), Eq5, and Eq8.

[0099]The present invention is also related to an antigenic composition,
wherein said at least one protein or polypeptide is selected from the
group consisting of EAG, Eq8, and IdeE2 and which composition comprises
at least one further antigen which is selected from the group comprising
IdeE, Eq5, IdeZ2, Eqz5 and Eqz8 and/or ScIC and CNE (or SEC).

[0100]According to the present invention, the antigenic composition
suitably comprises at least one antigen which is recombinantly produced
and/or at least one antigen which is an isolated or purified antigen.

[0101]From the above, it is evident that the present antigens or
immunogens that are derived from proteins of Streptococcus equi may
comprise the entire protein, a fragment of said protein or an analog of
said protein which is antigenic or immunogenic. Thus, the present
invention is not limited to the fragments of proteins that are
specifically disclosed herein.

[0102]The antigenic composition of the present invention may comprise at
least one recombinant vector and at least one polynucleotide inserted
therein that encodes said at least one protein or polypeptide, and which
vector is able to express said polypeptide in vivo in a non-human mammal
susceptible to infection with S. equi.

[0103]According to one embodiment of the present invention, the vector is
an expression vector which is a plasmid or a viral vector and wherein
said polynucleotide has a nucleotide sequence that encodes an antigen of
the present invention.

[0104]A further embodiment of the present invention is concerned with a
vaccine composition for protecting non-human mammals against infection of
Streptococcus equi, which comprises an antigenic composition as disclosed
above as immunizing component, and a pharmaceutically acceptable carrier.

[0105]Suitably, the present vaccine composition comprises an antigenic or
immunogenic composition that contains 2, 3 or more of the present
antigens or immunogens as immunizing components. Optionally, one or more
of these antigens or immunogens are comprised of analogs of said proteins
or fragments thereof, e.g. N-terminal or C-terminal fragments.

[0106]The vaccine composition may comprise further components, such as an
adjuvant. Suitably, said adjuvant stimulates systemic or mucosal
immunity. Such adjuvants are well known in the art.

[0108]A suitable adjuvant for use according to the present invention is
the adjuvant Abisco from Isconova AB, Sweden. The key components of
ISCOMS are Quillaia saponins derived from the bark of the chilean soap
bark tree Quillaia saporinaria molina. Quillaia saponins are well known
for their ability to activate the immune system. Quillaia saponins mixed
with cholesterol, and phospholipids under specific stoichiometry form
spherical open cage like structures known as ISCOMS.

[0109]Another suitable adjuvant is Ginseng. Ginseng is a dry extract
prepared from the root of the plant Panax ginseng, C. A. Meyer. Ginseng
contains a number of active substances named ginsenosides that are a kind
of saponins, chemically tri-terpenoid glycosides of the dammaran series.
The ginsenosides have adjuvant properties and one of the most active
adjuvant is the fraction named Rb1. It has been proved that the fraction
Rb1 elicits a balanced Th1 and Th2 immune response as determined by
measuring the levels of the cytokines IFN-γ, IL-2, IL-4, IL-10
secreted post vaccination with a Rb1 adjuvanted vaccine. In addition
ginseng and the fraction Rb1 stimulates a strong antigen specific
antibody response.

[0110]According to a suitable embodiment, the vaccine composition is a
vaccine that protects susceptible mammals, suitably horses, against
strangles caused by Streptococcus equi subsp. equi.

[0111]The vaccine composition of the present invention is provided in a
physiologically administrable form. Suitably, it is administrable by
subcutaneous, intramuscular or intranasal inoculation.

[0113]The present invention is also related to a method for producing an
antigen or immunogen to be used in an antigenic or immunogenic
composition of the present invention, which method comprises

[0114](a) providing a DNA fragment encoding said antigen and introducing
said fragment into an expression vector;

[0115](b) introducing said vector, which contains said DNA fragment, into
a compatible host cell;

[0116](c) culturing said host cell provided in step (b) under conditions
required for expression of the product encoded by said DNA fragment; and

[0117](d) isolating the expressed product from the cultured host cell.

[0118]Preferably, said method further comprises a step (e) wherein the
isolated product from step (d) is purified, e.g. by affinity
chromatography or other chromatographic methods known in the art.

[0119]Accordingly, the antigens of the present invention are usually
produced according to recombinant technique.

[0120]A further embodiment of the present invention is concerned with a
method for preparation of a vaccine of the present invention, which
vaccine contains as immunizing component an antigenic or immunogenic
composition as disclosed above, said method comprising mixing said
antigenic composition and a pharmaceutically acceptable carrier.

[0121]The present invention is also related to a method for the production
of an antiserum, said method comprising administering an antigenic
preparation of the present invention to an animal host to produce
antibodies in said animal host and recovering antiserum containing said
antibodies produced in said animal host.

[0122]Moreover, the present invention is concerned with a method of
prophylactic or therapeutic treatment of S. equi infection in non-human
mammals, suitably horses, comprising administering to said mammal an
immunologically effective amount of a vaccine or an antiserum of the
present invention.

[0123]Accordingly, the present invention is related to a method for
protecting horses against Streptococcus equi infection, which method
comprises inoculating a horse intramuscular, subcutaneously or
intranasally, or a combination of e.g. both subcutaneously and
intranasally, with a vaccine composition of the present invention to
induce an immune response against Streptococcus equi in said horse.
Suitably, an immune response, in the form of IgG and/or IgA and/or IgM
antibodies in the nasopharyngeal mucus, is induced in said horse.

[0124]The present invention also relates to an antibody preparation
comprising at least one, and suitably at least two, antibodies specific
for a protein or a polypeptide of the present antigenic composition,
which antibody/antibodies is/are polyclonal or monoclonal; or which
preparation comprises a fragment of said antibodies.

[0125]The antibody preparation of the present invention could be used
prophylactically or therapeutically against strangles and provides
passive immunization when administered to a non-human mammal susceptible
to infection by Streptococcus equi or infected by Streptococcus equi.

[0126]The present invention describes a vaccine composition comprising one
or several antigen components which have been prepared according to the
present method using E. coli as host cells. The source of these antigens
might also be the native bacteria, if methods are developed for
expression and purification thereof. Alternatively, the antigens of the
present invention can also be produced according to methods that are
based on fusion strategies where various parts of the respective antigen
are recombined resulting in a fusion can in protein consisting of parts
from different antigens. This fusion strategy could also be suitable for
introducing immune reactive part(s), e.g. T-cell epitopes or attenuated
toxins (or parts thereof), thereby introducing other features suitable
for optimizing the antigen presentation or localization. Furthermore,
other hosts for expressing the recombinant antigens addition to E. coli
also be other suitable species of bacteria and viruses. Today many
different systems for expression of heterologus expression are well known
in the field of molecular biology.

[0127]Yet another implication of this invention is that it can be used to
design specific attenuated mutants of S. equi that lack or have
inactivated genes important for survival (i.e. mutations causing
deficiency in metabolic pathways) in the host but retain or overproduce
the antigens of the present invention.

EXPERIMENTAL PART

[0128]The DNA sequence of the genome of S. equi subsp. equi and subsp.
zooepidemicus have been determined (www.sanger.ac.uk/) but not yet
annotated. By screening open reading frames a great number of genes
encoding extracellular proteins were identified. Among these genes a
selected number were chosen and recombinant proteins were produced and
evaluated in vaccine studies. The cloning and expression of these genes
is described below. Furthermore, the use of these proteins as antigens
will also be described.

[0129]Chromosomal DNA from S. equi subspecies equi strain 1866
(PCT/SE03/01587, Lannergard and Guss 2007) was used as a template to
amplify potential genes encoding IdeE2, Eq5 and Eq8 (the nucleotide- and
protein-sequences are presented in the sequence listing further below).
To identify the predicted signal sequences, the computer program SignalP
(http://www.cbs.dtu.dk/services/SignalP/) was used. The sequences of
primers used to amplify the genes or part of the genes ideE, ideE2, eq5
and eq8 are listed in the Primer Table. Cleavage sites for the
restriction enzymes NcoI and XhoI were included in the primer sequences
to match the cloning sites in the plasmid vector pTYB4 (New England
Biolabs). The PCR amplifications were performed using the primers (20
pmol/μl) and the ReadyToGo® PCR beads (GE Healthcare) using the
following programme: Step 1, pre-heat 1 minute at 95° C., DNA
strand separation; Step 2, 30 seconds at 95° C.; Step 3, annealing
15 seconds at 46° C.; and Step 4, elongation for 2 minutes at
72° C., Steps 2-4 were run for 26 cycles. The PCR products were
analysed on a 1 agarose gel, and thereafter purified using the QIAquick
PCR Purification Kit® (Qiagen). Cleavage with the restriction enzymes
was performed over night whereupon the fragments were purified one
additional time using the same kit.

Primer Table: The primer sequences used to PCR amplify the genes ideE,
ideE2, eq5 and eq8. The nucleotides underlined correspond to the
introduced restriction cleavage sites NcoI and XhoI.

To clone and produce recombinant proteins in E. coli the IMPACT®
Protein Purification System (New England Biolabs) was used. E. coli
strain ER2566 containing the pTYB4 vector (New England Biolabs) was grown
according to the manufacturer's instructions, and the vector was purified
using the QIAprep Spin Miniprep (Qiagen). Purified vector was digested
using restriction endonucleases NcoI and XhoI. After digestion, the
vector was treated with the enzyme alkaline phosphatase to reduce the
background of re-ligated vector in the later ligation step. For the
ligation of the vector and the respective PCR product, the ReadyToGo
T4DNA Ligase (GE Healthcare) was used. After ligation, the respective
sample were transformed into competent cells of E. coli strain ER2566
using electroporation, and spread on LA-Amp plates (Luria-Bertani broth
agar plates supplemented with ampicillin, final conc. 50 μg/ml) and
incubated over night at 37° C. Next day colonies were counted and
four colonies per construct were cultivated and used for further
experiments. To verify the presence of an insert in the respective
constructs, plasmids were purified and additional PCR analyses were
performed using the respective primer combination. The sequence of the
respective insert was also determined by DNA sequencing using primers
that hybridise in the vector (T7 universal forward primer and a reverse
primer located in the intein coding region).

[0130]Cloning of the ideE gene of S. equi subsp. equi strain 1866 has been
reported previously by Lannergard and Guss (2006). The GenBank accession
number of ideE is DQ508733. The part of the gene used to obtain the
recombinant IdeE protein used for immunization was cloned using the
primers IdEG1 and IdEG2 listed in the Primer Table. After PCR
amplification the DNA fragment was digested with restriction enzymes
BamHI and XhoI and ligated into the vector pGEX6-P-1 (GE Healthcare),
previously digested with the same enzymes.

Example 2

Preparation of Antigens CNE, ScIC, EAG4B, IdeE, IdeE2, Eq5 and Eq8

[0131]The vector used is a part of an E. coli expression and purification
system called IMPACT® T7 (NEB Inc.) Briefly, following the
manufacturer's instructions the clones expressing recombinant IdeE2, Eq5
and Eq8, respectively were grown at 37° C. in Luria Bertani growth
medium supplemented with ampicillin (final conc. 50 μg/ml). At an
optical density (OD600)˜0.6, the growth medium was
supplemented with IPTG (final conc. 0.4 mM) and the growth temperature
shifted to 20° C. After incubation over night the cells were
harvested and resuspended in a buffer [20 mM Tris-HCl (pH 8.0), 500 mM
NaCl, 0.1 mM EDTA, and 0.1% Triton X100] and lysed by freezing and
thawing. After centrifugation, the supernatant was sterile filtrated and
applied onto a chitin column. The columns were extensively washed using
the same buffer and subsequently treated with cleavage buffer [20 mM
Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, and 30 mM dithiothreitol
(DTT)]. In the cleavage buffer, the reducing conditions induce an
intein-mediated self-cleavage that releases the antigen part from the
column while the intein-chitin-binding part is still bound. The eluted
samples containing the antigens were dialysed against phosphate-buffered
saline [PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.4 mM
KH2PO4 (pH 7.4)] and concentrated. The amounts of antigens
obtained were determined and the quality was checked using SDS-PAGE. The
recombinant IdeE protein was produced and purified using the GST-affinity
chromatography system according to the procedure recommended by the
manufacturer (GE Healthcare). The description of and production of the
recombinant proteins CNE(SEC), ScIC, and EAG4B antigens have been
described previously (WO 2004/032957 (PCT/SE03/01587), Waller et al
2007). In the following examples, the EAG4B protein is simply called EAG.

Example 3

Recombinant IdE2 Cleaves IgG

[0132]IdE has previously been shown to be a protease that specifically
cleaves IgG from various species (Lannegard and Guss 2006). To test if
recombinant IdeE2 also cleaves antibodies, IgG from human, horse and
mouse were incubated in PBS at 37° C. for one hour. Purified
recombinant IdeE was used as a positive control and the negative control
was pure IgG. After cleavage, the samples were analysed using 8-25%
gradient SDS-PAGE. The result showed that recombinant IdeE2 cleaves
cleaves horse IgG much more efficiently than IdeE does.

[0133]Previously the presence of a homologous subsp. equi ideE gene in
subsp. zooepidemicus has been reported (Lannegard and Guss 2006). Using
the S. zooepidemicus genome database (www.sanger.ac.uk/), the presence of
similar genes to ideE2, eq5 and eq8 in subspecies zooepidemicus was
analysed using BLAST search. The results showed that genes encoding
similar proteins were detected. The sequence of these genes called ideZ2,
eqz5 and eqz8 along with amino acid sequences IdeZ2, Eqz5 and Eqz8 are
shown in the list of sequences in the experimental part of this
specification.

Example 5

Immunisation of Mice with Eq5 and Eq8

[0134]Mice (NMRI) weighting approximately 23-25 g were kept in cages of
five animals in each. The mice were immunised intranasally with 12
micrograms of each antigen and 10 microgram of Abisco 300 (Isconova AB,
Sweden). Fifteen animals were immunised with antigen (Eq5 and Eq8) and 15
were only given Abisco 300 adjuvant to serve as a negative control. The
total volume was kept to less than 27 μl and applied into the nostrils
twice with 30 minutes interval of mice anaesthetized with Isoflovet
(Abbot Laboratories, England). Immunisations were given on days 0, 13 and
32.

Example 6

Immunisation of Mice with EAG, IdeE and IdeE2

[0135]Immunisation with EAG, IdeE and IdeE2 was performed essentially as
for Eq5 and Eq8. However, animals were divided into three groups, with
ten mice in each group. These were given EAG+IdeE+IdeE2 or EAG only and
one group with only adjuvans, Abisco 300, as negative control.
Immunisations were given on days 0, 21 and 53. Experimental infection was
given on day 60.

Example 7

Experimental Infection with Streptococcus equi subsp. equi

[0136]Experimental infection was given on day 43 (10 days after last time
of immunisation) for Eq5+Eq8 and on day 60 (10 days after last
immunisation) for EAG+/-IdeE+IdeE2. S. equi subsp. equi strain 1866 from
a clinical case of strangles was used. The strain was first passed
through an animal by inoculating ca 106 CFU into the nostrils of an
anaesthetized mouse. Bacteria were recovered after 7 days from the nose
of the mouse and grown on BG plates at 37° C. in 5% CO2. A
single colony was grown on BG plates overnight at 37° C. and
resuspended in Todd Hewitt Broth (THB) with 1% yeast extract (THY). The
culture was kept at -80° C. in vials and a new vial was used for
each experiment. To infect mice, bacteria were grown on BG plates at
37° C. in 5% CO2 overnight, followed by inoculation into THY
and grown without shaking over night. The cultures was then diluted 10
times into THY and 10% horse serum (Sigma) and grown for 4 hours at
37° C. in 5% CO2. The culture was centrifuged and resuspended
in THB. A dose containing 1×106 CFU in 10 μl was used for
all S. equi infections of mice. The animals were followed daily.
Bacterial nasal growth was scored on a four-graded scale from 0 to +++ by
gently pressing the nose of the animal onto a blood agar plate in a
reproducible manner. The nasal sample was then spread out onto the entire
surface of the plate. One + means 5-100 colonies; two + means more than
100 and three + means confluent growth. The weight was determined every
day and the percentage of weight-loss was calculated.

Example 8

Experimental Results of Vaccination

[0137]Mice were immunised with both Eq5 and Eq8 and the percentage weight
loss over time was determined. FIG. 1 shows that vaccinated animals
(n=15) lost less weight that control animals (n=15). P-values=0.0001 for
all days (Student's t-test). Nasal growth of S. equi was also determined
daily on a four graded scale. FIG. 2 shows that the vaccinated animals
had much less nasal growth than the control group. The frequency of
animals grossly colonised nasally with bacteria (scoring 2-3) on day 5
was significantly different between the two groups; p=0.002 (Fisher's
exact test).

[0138]In the next experiment, mice were vaccinated with EAG (n=10), with
EAG+IdeE+IdeE2 (n=10) or non-vaccinated (n=10). The percentage weight
loss over time was determined. FIG. 3 shows that animals vaccinated with
EAG+IdeE+IdeE2 lost less weight that control animals. P values were
0.0013, 0.0008 and 0.0009 for days 3, 5 and 6 respectively (Student's
t-test). Animals vaccinated with EAG alone also lost weight to a similar
magnitude as control animals. Nasal growth of S. equi was also determined
daily on a four graded scale. FIG. 4 shows that the animals vaccinated
with EAG+IdeE+IdeE2 had much less nasal growth than the control group.
Again, vaccination with only EAG showed no protection.

Example 9

Immunisation of Mice with Eq5, Eq8, and EAG, CNE, ScIC

[0139]Immunisation i.n. with Eq5+Eq8 and EAG+CNE+ScIC was performed as
above with three groups with ten mice in each group. One group with
Eq5+Eq8 and one with EAG+CNE+ScIC. The third group was the control with
Abisco-300. Immunisations were given on days 0, 14 and 22. Challenge was
given on day 29. The experimental results are shown in FIG. 5a and FIG.
5b. FIG. 5a and b show significant protection for EAG+CNE+ScIC (n=10).
P-values were 0.04 and 0.09 for day 2 and 5. The protection with Eq5+Eq8
was even more pronounced where p-values were 0.005 and 0.009 for these
days.

(2) SEQ ID NO: 2 shows the recombinant IdeE2 protein sequence. The amino
acids in bold are those that corresponds to the amino acids encoded by
the pTYB4 vector while the rest originates from the IdeE2 protein.

(4) SEQ ID NO: 4 shows the recombinant Eq5 protein sequence: The amino
acids in bold are those that corresponds to the amino acids encoded by
the pTYB4 vector while the rest originates from the Eq5 protein.

(6) SEQ ID NO: 6 shows the recombinant Eq8 protein sequence: The amino
acids in bold are those that corresponds to the amino acids encoded by
the pTYB4 vector while the rest originates from the Eq8 protein.

[0147]The objective of this study was to determine the level of protection
conferred on vaccination with Intervacc's new multi-component subunit
vaccine following intranasal challenge with wild type S. equi strain 4047
in Welsh Mountain ponies. The study has been performed by Animal Health
Trust, UK. The vaccines used therein, which are designated Nordostrep
Septavac or Nordostrep Pentavac A (or only Septavac or Pentavac) are
disclosed below.

Methods

[0148]The ponies were initially randomised into 3 groups for the
vaccination period.

In the first trial groups 1 and 3 were taken through to challenge. (The
challenge of second trial group 2 (Pentavac A) is described in section
9). The decision as to which vaccine group to challenge was taken by
Intervacc one week prior to challenge.

The Nordostrep Pentavac A Formulation

[0149]The Pentavac vaccine consisted of the following five S. equi
recombinant proteins: EAG, ScIC, CNE, Eq5 and Eq8. For subcutaneous
vaccination, the five proteins were mixed in PBS (50 μg/ml of the
respective protein), divided in aliquots of 1 ml in vials and stored at
-20° C. Immediately before vaccination, the vial was thawed and
mixed with 1 ml adjuvant (Abisco 200, 375 μg/dose, Isconova AB,
Sweden). For intranasal vaccination the five proteins were mixed in PBS
(150 μg/ml of respective protein) and divided in aliquots of 2 ml in
vials and stored at -20° C. Immediately before vaccination the
vial was thawed and mixed with 2 ml adjuvant (Abisco 300, 500 μg/dose,
Isconova AB, Sweden). In the placebo formulations the S. equi proteins
were omitted. Thus, the placebo for subcutaneous vaccination only
contained PBS and Abisco 200, 375 μg/dose and for intranasal
vaccination, the placebo contained only PBS and Abisco 300, 500
μg/dose.

The Nordostrep Septavac Formulation

[0150]The Septavac vaccine consisted of the following seven S. equi
recombinant proteins: EAG, ScIC, CNE, Eq5, Eq8, IdeE and IdeE2. For
subcutaneous vaccination, the seven proteins were mixed in PBS (50
μg/ml of respective protein) and divided in aliquots of 1 ml in vials
and stored at -20° C. Immediately before vaccination the vial was
thawed and mixed with 1 ml adjuvant (Abisco 200, 375 μg/dose, Isconova
AB, Sweden). For intranasal vaccination, the seven proteins were mixed in
PBS (150 μg/ml of the respective protein) and divided in aliquots of 2
ml in vials and stored at -20° C. Immediately before vaccination,
the vial was thawed and mixed with 2 ml adjuvant (Abisco 300, 500
μg/dose, Isconova AB, Sweden). In the placebo formulations, the S.
equi proteins were omitted. Thus, the placebo for subcutaneous
vaccination only contained PBS and Abisco 200, 375 μg/dose, and for
intranasal vaccination, it only contained PBS and Abisco 300, 500
μg/dose.

[0151]In these formulations, EAG is comprised of the fragment EAG4B and
CNE is the fragment designated 2.16.

Short Summary of Results

[0152]This study evaluated the efficacy of a new multi-component subunit
vaccine for the prevention of strangles. The Septavac vaccine induced
pyrexia in ponies for one day after first and second vaccinations.
However, there were no other adverse reactions and this vaccine appears
to be very well tolerated.

[0153]All ponies were challenged with an identical dose of
1×108 cfu of S. equi strain 4047, which was split and
administered via both nostrils. All seven control ponies developed
pyrexia and multiple lymph node abscesses (100%). Only one vaccinated
pony developed pyrexia (which could have been due to an ongoing S.
zooepidemicus infection) and only one developed lymph node abscesses
(14%). Statistically, vaccinated ponies were significantly protected from
S. equi as measured by temperature, post mortem score, and fibrinogen and
neutrophil levels.

[0154]Overall, the Septavac vaccine was a safe and effective vaccine for
the prevention of strangles. However, the invention is not restricted to
the Septavac and Pentavac vaccines which have been studied in this
Example but many combinations of the present antigens/immunogens are
possible candidates for use in vaccine compositions for prevention of
strangles.

1 Procedure

[0155]Two earlier studies (WO 2004/032957 A1 and ref. 27) demonstrated
that Intervacc vaccines conferred some protection against S. equi
challenge. All four vaccinated groups across the two studies showed
reduced guttural pouch empyema. The present study was designed to compare
the immunogenicity of two Nordvacc vaccines: one containing five
(Pentavac) and one containing seven (Septavac) S. equi proteins.

[0156]Blood and nasal wash samples were taken according to the protocol to
determine the equine immune responses to the vaccine subunits. Based on
immunogenicity data, one vaccinated group was challenged to quantify the
level of protection conferred.

[0157]Each pony was challenged with a total challenge dose of
1×108 cfu of S. equi strain 4047 administered via the spraying
of a 2 ml culture containing 5×107 cfu into both nostrils.
This dose regime is believed to optimise the infection rate whilst
avoiding overwhelming the host immune response.

[0158]Ponies were carefully monitored for the onset of clinical signs of
disease over a period of three weeks post challenge by regular checks,
daily physical examination, monitoring of body temperature, the taking of
sera to determine seroconversion and the taking of nasal washes for
bacteriological analysis. All ponies were subjected to post mortem
examination following abscessation or reaching the study endpoint at 3
weeks post challenge to determine the severity of disease pathology
according to a scoring system developed at the AHT. Histopathological
examination of tissues recovered from the study ponies was used to
identify early signs of S. equi infection that were not obvious on post
mortem (PM) examination.

[0172]The vaccine vials were received by the AHT prior to the first
vaccination and stored at -20° C. until use in freezer number EQ
No. 2305. Placebo (containing no antigens) and adjuvant vials were stored
at 4° C. until use in fridge number EQ No. 44.

[0173]At the time of vaccination, vaccines and adjuvants were mixed as
stated in the protocol in situ by A Waller, L Prowse or C Robinson at
AHT.

2.2 Challenge Bacterium

[0174]S. equi 4047 was prepared from fresh plates as described in
SOP/BACT/25.

[0185]Prior to challenge, ponies were kept at pasture on grass at Lanwades
Park, Kentford, UK and Kirtling, Newmarket, UK. These sites have been
approved by the Home Office for this type of work. Drinking water was
available ad libitum.

[0186]Ponies in groups 1 and 3 were transferred to the ACVS (Allen
Centre), three days prior to challenge to allow acclimatisation. Ponies
were separated into two animal rooms according to their vaccination
groups, so that ponies from each vaccination group were kept together.

Methods

4.1 Vaccination

[0187]Vaccinations were given by subcutaneous injection near the
retropharyngeal lymph nodes according to AHT SOP/EQU/03 or via intranasal
spray according to AHT SOP/EQU/07.

4.1.2 Preliminary Clinical Examination

[0188]A veterinarian clinically examined all ponies before the first
vaccination, before V2 (due to S. zoo infection) and before V3. Only
healthy ponies in good clinical condition were included in the study
(SOP/EQU/08).

4.1.3 Vaccination

[0189]Ponies received vaccinations according to Table 4. With the
exception that pony 9229 was pyrexic on Feb. 14, 2008 due to an ongoing
S. zooepidemicus infection. This pony recovered over the weekend and was
vaccinated on Apr. 18, 2008.

[0190]Clinical observations were performed daily after vaccination. If
adverse reactions occurred, then additional checks were made as required.

4.2 Experimental Challenge with S. equi 4047

4.2.1 Preliminary Clinical Examination

[0191]Prior to transfer to the ACVS, a veterinarian clinically examined
the challenge ponies. Only healthy ponies in good clinical condition were
subjected to the challenge.

4.2.2 Challenge

[0192]Two weeks after the third vaccination (Aug. 5, 2008), each pony was
challenged by intranasal administration of 2 ml of a fresh S. equi 4047
culture into both nostrils using a flexible tube and spray nozzle
according to AHT SOP/BACT32. Such a challenge dose was predicted to
contain a total of 1×108 cfu of S. equi 4047.

[0193]No problems were encountered during the administration of the
challenge dose. Spare inocula were used to quantify the actual challenge
dose administered, which was found to be 1.37×108 cfu/dose.

4.3 Post Challenge Monitoring

4.3.1 Clinical Examination

[0194]Ponies were examined according to AHT SOP/EQU/02. Each pony was
examined clinically on the day of challenge, and on each of the following
21 days for the occurrence of symptoms associated with S. equi infection
(demeanor, nasal discharge, lymph node swelling and abscessation, signs
of coughing, difficulty swallowing and feeding, and ocular signs).

4.3.2 Rectal Temperatures

[0195]Individual rectal temperatures were taken at around 9.00 am from the
day of challenge through to day 21 after challenge.

4.4 Blood Sampling

[0196]Blood samples were taken from the jugular vein according to AHT
SOP/EQU/01 and according to the study protocol schedule. Serum was
prepared according to AHT SOP/EQU/01 and stored frozen at -20° C.
or below until use.

4.5 Processing of Blood Samples

[0197]Processing of blood samples was carried out by Leah Prowse under the
responsibility of Andrew Waller at the Animal Health Trust.

4.6 Processing of Nasal Wash Samples

[0198]Individual nasal washes were taken according to AHT SOP/EQU/02 as
stated in the study protocol schedule.

[0199]A 500 μl sample of the nasal wash was added to 500 μl of
Todd-Hewitt Broth in situ at the time of sampling for transportation to
the lab to allow quantification of the number of β-haemolytic
streptococci per ml according to AHT SOP/BACT/02. The remaining nasal
wash sample was centrifuged and the supernatant decanted into a clean 5
ml polypropylene tube and stored at

-70° C. until use for quantification of mucosal antibodies.

4.7 Post Mortem Examination

[0200]Provision was made for a complete post mortem examination to be
carried out by the Animal Health Trust on all ponies following euthanasia
as a result of abscessation or on reaching the study end point 21 days
post challenge.

[0201]Tissue samples were preserved in phosphate buffered formalin and
subjected to microscopic examination according to standard techniques and
provision of a full and formal report. Tissue swabs were taken and the
results recorded and used to evaluate the level of S. equi infection.
Charcoal swabs were taken from each of the areas as stated in the
protocol and processed on COBA Streptococcal selective plates to
determine the presence of S. equi.

[0203]Tissue samples taken from ponies at post mortem examination were
fixed in formalin, cut into sections and sent to Professor Ken Smith at
the Royal Veterinary College for analysis. Professor Smith prepared a
report for the samples from each pony and his observations were scored
according to Table 6.

[0204]The study was performed in accordance with the study protocol no.
08.C001.P and subsequent amendments, with the following deviations from
the agreed study protocol: [0205]Pony 9229 was pyrexic on Feb. 14, 2008
due to an ongoing S. zooepidemicus infection. This pony recovered over
the weekend and was vaccinated on Apr. 18, 2008. [0206]Date of V2 delayed
7 days due to S. zoo infection in 45% of ponies. This had a knock on
effect on V3 and challenge which were also delayed 7 days. [0207]A delay
of one day occurred on sampling ponies due to staff shortages. Ponies due
to be sampled on day 85 were actually sampled on day 86. [0208]20 ml of
EDTA blood was taken on day 86 instead of 10 ml to enable purification of
the ponies' DNA for archiving. [0209]Nordvacc decided to retain the
unchallenged Pentavac group (2) for a 6-month period to monitor the
duration of antibody response.

6 Fate of Ponies at the End of the Study

[0210]All ponies in groups 1 and 3 were euthanased and subjected to post
mortem examination. Ponies in group 2 were retained for 6 months to
monitor the duration of antibody responses.

[0212]All ponies responded well to first vaccination. No injection site
reactions were observed in any of the groups. However, a rise in rectal
temperature was observed in the vaccinated groups (FIG. 7). This was most
pronounced in the Septavac group with 4 of 7 ponies developing pyrexia
(temperature>38.9° C.) one day post V1 and 5 of 7 ponies
developing pyrexia one day post V2. In comparison, 2 of 7 ponies of the
Pentavac group and none of the controls were pyrexic post V1, and 3
ponies of the Pentavac group and no controls were pyrexic post V2.
Interestingly, only 1 Septavac, 2 Pentavac and 1 control pony developed
pyrexia post V3. This could be due to the high level of antibodies
induced post V2, which may have neutralized the antigens in the vaccine
more effectively.

[0213]There were no obvious differences in nasal score (FIG. 8), lymph
node score (FIG. 9) or S. zooepidemicus counts (FIG. 10) between the
study groups during the vaccination phase, with the exception of some
ponies that had ongoing S. zooepidemicus infections typical of ponies of
this age. This resulted in a rise in mean rectal temperature around the
original date for V2 (Apr. 3, 2008) as demonstrated in FIG. 7. Ponies
were allowed to recover from this S. zooepidemicus infection and all
ponies were vaccinated on Apr. 10, 2008.

8.2 Responses Following Challenge

[0214]The preparation and conduct of both challenges went extremely well
and all ponies received the required dose of S. equi without incident on
the May 8, 2008.

[0215]Earliest onset of pyrexia was at day 4 post challenge in control
pony 2078. Two more ponies developed pyrexia on day 5, another on day 6
and 7 and the final control pony developed pyrexia on day 10 (FIG. 11).
The mean number of days that control ponies were pyrexic was 4.2 days
compared with 0.7 days for vaccinated ponies (Table 7). However, it
should be noted that control ponies were euthanased on welfare grounds
from day 8 post challenge and all control ponies had been euthanased by
day 13 post challenge. This has had a knock on effect on the mean
temperatures, observation scores, fibrinogen and neutrophil levels and
observation scores for control ponies, which decline as ponies succumbing
to S. equi infection were euthanased.

[0216]Overall, there was a significant difference in the mean temperatures
of the two groups from day 5 to day 11 post challenge (FIG. 11). Of the
Septavac ponies only pony 0976 developed pyrexia on day 8 (Table 7).
However, this may have been due to the ongoing S. zooepidemicus infection
that was evident in this pony.

[0217]Fibrinogen levels were significantly different between the two study
groups on days 6, 8 and 11 post challenge (FIG. 12). All controls
developed elevated fibrinogen levels, but only 2 vaccinates (ponies 0976
and 9794) had higher levels.

[0218]Neutrophil levels were also significantly different between the two
study groups on days 6, 8 and 11 post challenge (FIG. 13). All controls
developed elevated neutrophil levels, but only 1 vaccinate (pony 9794)
had higher levels.

[0219]There was an increased level of submandibular lymph node swelling in
control ponies, although this did not appear to be statistically
significant (FIG. 14). There were no differences in nasal discharge (FIG.
15) or S. zooepidemicus counts (FIG. 16) between the study groups.

[0220]On post mortem examination, all controls were found to have multiple
lymph node abscesses, while only one vaccinated pony, 9794, was found to
have lymph node abscesses (Tables 8 and 9). Overall the mean pathology
score for controls and 11.7, respectively indicating that a significant
level of protection had been induced by the Septavac vaccine (FIG. 17).
S. equi was isolated from the lymph nodes of all control ponies, but only
2 vaccinates (0976 and 9794) (Table 10). These findings were strengthened
by histopathological examination, which confirmed that only one Septavac
pony had developed abscesses in at least two of their lymph nodes (Table
11 and FIG. 18).

[0221]Furthermore, the IgG levels in nasal washings and serum samples of
the septavac group were measured using ELISA (FIGS. 19 and 20) showing
that the antigens generate mucosal and serum antibodies.

TABLE-US-00024
TABLE 7
Number of days pyrexic after challenge
Pony Number of
Group ID days
Septavac 0012 0
Septavac 0159 0
Septavac 0833 0
Septavac 0976 5
Septavac 9123 0
Septavac 9668 0
Septavac 9794 0
Control 0173 2
Control 0427 4
Control 1635 5
Control 2078 4
Control 9549 4
Control 9776 5
Control 9886 6
Mean Septavac = 0.7 days
Mean control = 4.2 days*
*All control ponies were euthanased by day 13 post-challenge, but most
would have continued to have elevated temperatures had they not been
euthanased on welfare grounds.

[0224]In the second trial the seven horses of group 2 (section 3.2, table
3) where after vaccination V3 (Table 4) kept at pasture on grass and
blood samples where taken regularly to measure IgG antibody titers in
ELISA against the five antigens present in the Pentavac A formulation
(FIG. 21). In Day 270 (Nov. 6, 2008) a booster dose of Pentavac A was
given according to the procedure described in section 4.1. Before
challenge the group was transferred to ACVS and fourteen days post
booster the group was experimentally challenged with S. equi 4047 as
described in section 4.2 and monitored essentially as described in
section 4.3.

9.1 Brief Summary of the Pentavac A Vaccination Study

[0225]The Pentavac A study revealed that after vaccination a significant
antibody response against the individual antigens remains for at least
six months (FIG. 21).

The Pentavac A vaccine delayed the onset of infection upon challenge with
S. equi and that one of the ponies in the group did not developed
strangles.

Further Applications

[0226]One implication of the present invention is that enzymes degrading
immunoglobulins can be used as antigens in a vaccine to protect the
target animal from infection. Therefore one embodiment of the present
invention is that concerning the human pathogenic group A streptococci
(GAS) it is possible to construct a vaccine composition which protects
humans from infections caused by this bacterium. In strains of GAS there
are several reported extracellular immunoglobulin degrading proteins
(called Sib35, IdeS or Mac-proteins) which share amino acid sequence
homologies to IdeE and IdeE2 and therefore in light of the present
invention can be purified and used as antigens in a vaccine separately or
in combination with other purified extracellular proteins (like
M-proteins or M-like proteins or fragments thereof) from group A strains.
As in the present invention another implication is that the invention can
be used to develop specific antisera, polyclonal or monoclonal antibodies
to be used for diagnostic purposes or to be used in passive immunisations
of the target animal including humans.